The present invention is concerned with treatment of autoimmune diseases. The invention involves an agent such as a humanised monoclonal antibody that may be administered to patients in higher dosages than previously described. It is particularly effective for patients having diseases or characteristics requiring higher doses for effective treatment. The invention envisages a pharmaceutical composition comprising the agent, such as the antibody, in efficacious concentration, as well as uses and methods of treatment employing the compositions and medicaments comprising the agent.
Autoimmunity is the failure of an organism to recognise its own constituent parts (down to sub-molecular levels) as “self”, which results in an immune response against its own cells and tissues. Any disease that results from such an aberrant immune response is termed an autoimmune disease. Autoimmune diseases include multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, colitis ulcerosa, Crohn's disease, myasthenia gravis (MG), autoimmune polyglandular syndrome type II (APS-II), Hashimoto's thyreoiditis (HT), type-1 diabetes (T1D), systemic lupus erythematosus (SLE) and autoimmune lymphoproliferative syndrome (ALS).
Autoimmune disease occurs when T cells recognise and react to ‘self’ molecules, that is, molecules produced by the cells of the host. Activation of ‘autoreactive’ T cells by presentation of autoantigens processed by antigen presenting cells (APC) leads to their clonal expansion and migration to the specific tissues, where they induce inflammation and tissue destruction.
Normally, T cells are tolerant with regard to autologous tissue and only react on presentation of heterologous structures. Central tolerance and peripheral tolerance comprise the two mechanisms by which the immune system hinders autoreactive T cells from inducing their deleterious functions. Central tolerance is mediated through negative selection. This process entails the elimination, through clonal deletion of autoreactive T cells, during ontogenic development in the thymus.
Peripheral tolerance is the backup available if central tolerance fails and autoreactive cells escape the thymus. This mechanism of tolerance occurs continuously throughout life, keeping autoreactive cells in check through immune ignorance (anergy), peripheral deletion and/or active suppression.
T regulatory cells (Tregs, formerly also designated “suppressor cells”) as part of active suppression maintain peripheral tolerance and regulate autoimmunity (Suri-Payer et al., J. Immunol. 157: 1799-1805 (1996); Asano et al., J. Exp. Med. 184:387-396 (1996); Bonomo et al., J. Immunol. 154: 6602-6611 (1995); Willerford et al., Immunity 3: 521-530 (1995); Takahashi et al., Int. Immunol. 10: 1969-1980 (1998); Salomon et al., Immunity 12: 431-440 (2000); Read et al., J. Exp. Med. 192: 295-302 (2000). In general, regulatory T cells inhibit the activation and/or function of T helper type 1 (TH1) and TH2 effector cells. Dysregulation in Treg cell frequency or functioning can lead to debilitating autoimmune diseases (Baecher-Allan et al., Immunol. Review 212: 203-216 (2006); Shevach, Annu. Rev. Immunol. 18: 423-449 (2000); Salomon et al., Immunity 12: 431-440 (2000); Sakaguchi et al., Immunol. Rev. 182: 18-32 (2001)).
Several subsets of regulatory T cells have been characterized. The family of Tregs consists of two key subsets: naturally arising e.g. CD4+CD25+ Tregs and peripherally induced, Tr1 and Th3 Tregs. Furthermore NKTregs and CD8+ Tregs have been described in humans and rodents (Fehérvari et al., J. Clin. Investigation 114: 1209-1217 (2004)).
Thymus-derived Treg cells (naturally occurring CD4+CD25+Treg) are the main regulatory cells involved regulating autoimmunity or pathogenic immune responses.
i) they are CD4+ T cells and constitute 5-10% of peripheral CD4+ T cells
ii) they mature in the thymus
iii) they are generally characterized by the combined expression of the IL-2 receptor (CD25), the low molecular isoform of the CD45 molecule, CD152 (CTLA-4), and the transcription factor FoxP3.
The role of Tregs is exemplified best by experiments involving reconstitution of immunodeficient nude mice with CD4+ cells that were depleted of CD25+ cells. CD4+CD25− reconstituted nude mice develop various organ-specific autoimmune diseases, such as gastritis, oophoritis, orchitis, and thyreoiditis (Suri-Payeret al.; J. Immunol. 160: 1212-1218 (1998)).
Inclusion of the CD4+CD25+ subset into reconstitution experiments with nude mice prevents the onset of these diseases (Sakaguchi et al., J. Immunol. 155: 1151-1164 (1995)). The protective value of CD4+CD25+ cells against organ-specific autoimmunity has also been shown in several other models of autoimmunity (e.g. autoimmune gastritis, prostatitis, oophoritis, glomerulonephritis, epidimytis and thyreoiditis) caused by neonatal thymectomy performed 3 days after birth (d3Tx) or inflammatory bowel disease caused by reconstitution of SCID mice with CD45RBhigh, CD4+CD25− T cells. Administration of anti-CD25 antibody in vivo in mice also induces organ-localised autoimmune disease.
The discovery of the importance of the transcriptional regulator FoxP3 in mouse CD4+CD25+ T regulatory cell function and the previous observations that patients with IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance), a severe inflammatory disease similar to that seen in mice deficient in CD4+CD25+ regulatory cells (scurfy syndrome), have mutations in FoxP3, provided a direct correlation between an autoimmune animal model, mouse regulatory T cells, and a human autoimmune disease (Sakaguchi et al., J. Immunol. 155: 1151-1164 (1995)).
The pharmaceutical mechanism of regulatory T cells is not fully clear. CD4+CD25+ Tregs inhibit polyclonal and antigen-specific T cell activation. The suppression is mediated by a cell contact-dependent mechanism that requires activation of CD4+CD25+ Tregs via the TCR but Tregs do not show a proliferative response upon TCR activation or stimulation with mitogenic antibodies (anergic) (Shevach, Nature Rev. Immunol 2: 389 (2002). Once stimulated, they are competent to suppress in an antigen-independent manner the response of CD4+ T cells and CD8+ T cells as well as inhibit B-cell activation and their clonal expansion.
There are additional data indicating that suppressor activity of CD4+CD25+ Tregs partially also relies on anti-inflammatory cytokines like TGF-β (Kingsley et al., J. Immunol. 168: 1080 (2002); Nakamura et al., J. Exp. Med. 194: 629-644 (2001)). The functional significance of TGF-β secretion is furthermore supported by the findings that TGF-β-deficient mice develop autoimmune disease and that administration of neutralizing antibodies to TGF-β abrogates in vivo the prevention of autoimmunity or tolerance-inducing activity of CD4+ T cells in some models.
Within the CD4+ T cell subset at least 2 more different types of cells with suppressive function may exist, which are induced after exposure to specific, exogenous antigen (called ‘adaptive or inducible regulatory T cells’): Type 1 T regulatory (Tr1) cells and Th3 cells. These cell types appear to be distinguishable from CD4+CD25+ Tregs based on their cytokine production profiles. However, the relationship between these different types is unclear and the modes of action are overlapping.
Tr1 cells were induced by repetitive stimulation of TCR in the presence of IL-10 and were shown to mainly down-regulate immune responses via the production of high levels of IL-10 together with moderate amounts of TGF-β (Chen et al., J. Immunol. 171: 733-744 (2003)).
Th3 cells (identified in a model of EAE after oral delivery of antigen) produce high amounts of TGF-β and variable amounts of IL-4 and IL-10. IL-4, itself, was shown to be a key factor for the differentiation of Th3 cells, in contrast to Tr1 cells that are differentiated with IL-10 (Chen et al., Science 265:1237-1240 (1994)).
Suppression of T cell function by using immunosuppressive drugs is a principal therapeutic strategy that has been used successfully to treat autoimmune diseases. However these drugs induce a general immunosuppression due to their poor selectivity, resulting in inhibition of not only the harmful functions of the immune system, but also useful ones. As a consequence, several risks like infection, cancer and drug toxicity may occur.
Agents interfering with T cell function are therapeutic mainstays for various autoimmune diseases.
The approach of using agents aiming at the activation of regulatory T cells for the therapy of autoimmune diseases have been up to now proven to be extremely difficult. Activation of Tregs via the TCR using the agonistic anti-CD3 antibody OKT-3 (Abramowicz et al, N Engl. J. Med. 1992 Sep. 3; 327(10):736) or via the co-stimulatory molecule CD28 using the superagonistic anti-CD28 antibody TGN 1412 leads to complete depletion of the regulatory T cell population as well as other conventional T cells and the systemic induction and release of excessive amounts of pro-inflammatory cytokines including IFN-γ, TNF-α IL-1 and IL-2, resulting in a clinically apparent cytokine release syndrome (CRS) in humans (Suntharalingam et al, N Engl. J. Med. 2006 Sep. 7; 355(10):1018-28).
After the first two to three injections of 5 mg of the monoclonal antibody OKT3 most patients develop a cytokine release syndrome with high levels of tumour necrosis factor-alpha, interleukin-2, and gamma-interferon appearing within 1-2 hrs in the circulation of kidney transplant recipients. (Abramowicz et al., Transplantation. 1989 April; 47(4):606-8). This results in a narrow therapeutic window which limits the usefulness of this antibody in the treatment of autoimmune disease.
Treatment with a total dose of 5-10 mg of TGN1412 (0.1 mg anti-CD28 per kilogram of body weight) lead to a systemic inflammatory response with multiorgan failure within 90 minutes after receiving a single intravenous dose of the TGN 1412 (Suntharalingam et al, N Engl. J. Med. 2006 Sep. 7; 355(10):1018-28).
It is generally agreed that CD4 T cells play a major part in initiating and maintaining autoimmunity. Accordingly, it has been proposed to use mAbs against CD4 T cells surface molecules, and in particular anti-CD4 mAbs, as immunosuppressive agents. Although numerous clinical studies confirmed the potential interest of this approach, they also raised several issues to be addressed in order to make anti-CD4 mAbs more suitable for use in routine clinical practice.
Several different mechanisms of action for CD4 mAbs have been proposed including: (1) antagonism of CD4-MHC II interactions resulting in inhibition of T cell activation, (2) CD4 receptor modulation as determined by a decrease in cell surface expression of CD4, (3) partial signaling through the CD4 receptor in the absence of T cell receptor cross-linking which can suppress subsequent T cell activation and trigger CD4 T cell apoptotic death, (4) Fc-mediated complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) leading to CD4 T cell depletion, and (5) stimulation of regulatory T cells.
Fc-mediated complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) leading to CD4 T cell depletion is the main observed mechanism and is especially demonstrated for antibodies of the IgG1 subclass. Only a few CD4 antibodies have been attributed to the other mechanisms like TRX-1, TNX-355, IDEC-151, OKTcdr4A with only TRX-1 being an IgG1 (Schulze-Koops et al., J Rheumatol. 25(11): 2065-76 (1998); Mason et al., J Rheumatol. 29(2): 220-9 (2002); Choy et al., Rheumatology 39(10): 1139-46 (2000); Herzyk et al., Infect Immun. 69(2): 1032-43 (2001); Kon et al., Eur Respir J. 18(1): 45-52 (2001); Mourad et al., Transplantation 65(5): 632-41 (1998); Skov et al., Arch Dermatol. 139(11): 1433-9 (2003); Jabado et al., J. Immunol. 158(1): 94-103 (1997)).
Dose-dependent depletion of CD4+ T cells at “high” doses (multiple cycles with dosages >100 mg) and transient sequestration (short-lived depletion) at “lower” doses (multiple cycles with dosages >10 mg), is observed with several CD4 antibodies (Mason et al., J. Rheumatol. 29 (2): 220-229 (2002); Kon et al., Eur. Respir J. 18(1):45-52 (2001)) and HuMax-CD4 (Skov et al., Arch Dermatol. 139(11): 1433-1439 (2003), Choy et al., Rheumatology 41 (10):1142-8 (2002)). Despite of their depletional activity, mAbs to CD4 failed to provide clinical benefit and consistent efficacy in autoimmune diseases investigated e.g. rheumatoid arthritis (Strand et al., Nature Reviews 6: 75-92 (2007)). Furthermore depletion of CD4+ T cells is generally considered as scenario, which might cause a severe immunosuppression.
The B-F5 antibody (murine IgG1 anti-human CD4) was tested in different autoimmune diseases.
A small number of patients with severe psoriasis have been treated with the murine B-F5 antibody and some positive effects were described (Robinet et al., Eur J Dermatol 1996: 6: 141-6, and Robinet et al., J Am Acad Dermatol 1997; 36: 582-8).
In rheumatoid arthritis patients, the results observed in a placebo controlled trial with a daily dose of B-F5 did not indicate a significant improvement (Wendling et al. J Rheumato 1;25(8): 1457-61, 1998).
In multiple sclerosis (MS) patients, some positive effects were observed after a 10 days treatment in patients with relapsing-remitting forms, some of who were still relapse-free at the 6th month post-therapy (Racadot et al., J Autoimmun, 6(6):771-86, 1993). Similar effects were observed by Rumbach et al. (Mutt Scler; 1(4):207-12, 1996).
In severe Crohn's disease, no significant improvement was observed in patients receiving B-F5 for 7 consecutive days (Canva-Delcambre et al., Aliment Pharmacol Ther 10(5):721-7, 1996).
In prevention of allograft rejection, it was reported that B-F5 bioavailability was not sufficient to allow its use for prophylaxis of allograft rejection (Dantal et al. Transplantation, 27;62(10):1502-6, 1996).
It appears from the above that a first issue to be solved is the need for using high doses of mAb to obtain a clinical improvement. This may result inter alia from the poor accessibility of the lymphocytes to the mAb in the target tissues. The use of higher doses may result in an excessive action on blood lymphocytes, inducing unwanted side effects.
Another drawback of therapy with monoclonal antibodies in humans is that these antibodies are generally obtained from mouse cells, and provoke anti-mouse responses in the human recipients. This not only results in a lower efficiency of the treatment and even more of any future treatment with mouse monoclonal antibodies, but also in an increased risk of anaphylaxis.
This drawback can, in principle, be avoided by the use of humanized antibodies, obtained by grafting the complementarity-determining regions (CDRs) of a mouse monoclonal antibody, which determine the antigen-binding specificity, onto the framework regions (FRs) of a human immunoglobulin molecule. The aim of humanization is to obtain a recombinant antibody having the same antigen-binding properties as the mouse monoclonal antibody from which the CDR sequences were derived, and far less immunogenic in humans.
In some cases, substituting CDRs from the mouse antibody for the human CDRs in human frameworks is sufficient to transfer the antigen-binding properties (including not only the specificity, but also the affinity for antigen). However, in many antibodies, some FR residues are important for antigen binding, because they directly contact the antigen in the antibody-antigen complex, or because they influence the conformation of CDRs and thus their antigen binding performance.
Thus, in most cases it is also necessary to substitute one or several framework residues from the mouse antibody for the human corresponding FR residues. Since the number of substituted residues must be as small as possible in order to prevent anti-mouse reactions, the issue is to determine which amino acid residue(s) are critical for retaining the antigen-binding properties. Various methods have been proposed for predicting the more appropriate sites for substitution. Although they provide general principles that may be of some help in the first steps of humanization, the final result varies from an antibody to another. Thus, for a given antibody, it is very difficult to foretell which substitutions will provide the desired result.
Previously the humanization of mouse B-F5 has been attempted, and success has been achieved in producing humanized B-F5 (hereinafter referred to as hB-F5) having similar CD4 binding properties to the parent mouse B-F5.
Thus, in WO 2004/083247, the humanised antibody BT061 (humanised B-F5, or simply hB-F5) has been found to be useful in treating autoimmune diseases, such as psoriasis and rheumatoid arthritis. This patent application discloses compositions for parenteral administration, formulated to allow the administration of a dose of from 0.1-10 mg, preferably from 1-5 mg. Dosage regimes envisaged are an intravenous 1 mg per day dose and a 5 mg every second day dose for rheumatoid arthritis patients over a period of 10 days. Thus the largest dose disclosed is 5 mg at one time and 25 mg over the course of 10 days.
The study was also described by Wijdenes et al., in an abstract and poster presented at the EULAR conference, June 2005. The treatment of 11 patients suffering from rheumatoid arthritis was disclosed. The patients were treated with 5 intravenous infusions of 5 mg BT061 every other day with concomitant treatment with 150 mg Diclophenac.
The antibody described in this study is not disclosed to be suitable for use in higher doses, and it is still desirable to find treatments at higher doses so as to treat a greater number of patients.